1. Field Of The Invention
The invention relates to a new microbiological process for hydroxylating methyl groups in aromatic 5- or 6-atom heterocycles as well as to new hybrid plasmids and new production strains especially suited for the process.
2. Prior Art
A microbiological process for the terminal hydroxylation of aliphatic side chains by genetically changed microorganisms is known from European Published Patent Application No. 0277674. This reaction is catalyzed by the alkane hydroxylase, coded by genes alkBA from the OCT-plasmid of Pseudomonas oleovorans. These microorganisms were changed genetically so that they are no longer capable of further oxidizing the resulting hydroxyl groups to the acid. But the natural expression and regulation (alkR) of these genes were maintained. These microorganisms have no activity for the oxidation of methyl groups in heterocycles, but catalyze only the hydroxylation of alkanes and alkylated compounds with alkyl radicals with 6 to 12 carbon atoms.
Further, it is known from Harayama et al., J. Bacteriol. 171, (1989), pages 5048 to 5055, that microorganisms of the species Pseudomonas putida with plasmid pWWO can oxidize the methyl group on toluene in three steps to benzoic acid. By the action of xylene monooxygenase (xylMA), benzyl alcohol first results, which in two further steps is then catalyzed by an alcohol dehydrogenase (xylB) and converted by an aldehyde dehydrogenase (xylC) to the acid. Both the xyl genes, which code for the enzymes of the xylene catabolism, and the genes which are responsible for the regulation of the xyl genes on plasmid pWWO, are in this strain. Thus, the properties, the identification, the cloning, the selection and the restriction map of the genes xylMABCN responsible for the oxidation of the methyl group are known from it. The function of gene xylN is still unknown. But no microbiological process is known which can hydroxylate methyl groups in aromatic 5- or 6-atom heterocycles. Moreover, those specifically hydroxymethylated heterocycles are chemically difficult to obtain.